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Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells
Published on: June 21, 2012
SWAP-70 contributes to spontaneous transformation of mouse embryo fibroblasts
Yu-Tzu Chang1, Chung-Li Shu1, Jing-Yang Lai1
1Institute of Cellular and System Medicine National Health Research Institute, Zhunan Town 35053, Miaoli County, Taiwan, ROC.
Abstract:
Mouse embryo fibroblasts (MEFs) grow slowly after cultivation from animals, however, after an extended period of cultivation, their growth accelerates. We found that SWAP-70 deficient MEFs failed to increase growth rates. They maintain normal growth rates and proliferation cycles for at least 5 years. Complementing SWAP-70 deficiency in one of these MEF clones, MEF1F2, by expressing human SWAP-70 resulted in fast growth of the cells after further cultivation for a long period. The resulting cells show a transformation phenotype, since they grow on top of each other and do not show contact inhibition. This phenotype was reverted when sanguinarine, a putative SWAP-70 inhibitor, was added. Two SWAP-70 expressing clones were examined in detail. Even after cell density became very high their cdc2 and NFκB were still activated suggesting that they do not stop growing. One of the clones formed colonies in soft agar and formed tumors in nude mice. Lately, one more clone became transformed being able to make colonies in soft agar. We maintain 4 human SWAP-70 expressing MEF1F2 cell lines. Three out of 4 clones exhibited transforming phenotypes. The mouse SWAP-70 gene also promoted transformation of MEFs. Taken together our data suggest that SWAP-70 is not a typical oncogene, but is required for spontaneous transformation of MEFs.
Insights
SWAP-70 deficiency in mouse embryo fibroblasts (MEFs) prevents accelerated growth. Expressing SWAP-70 in MEFs promotes cell transformation and tumor formation, indicating its role in spontaneous transformation.
Area of Science:
- Cell Biology
- Molecular Biology
- Oncology
Background:
- Mouse embryo fibroblasts (MEFs) typically exhibit slow initial growth that accelerates over time.
- SWAP-70 (SRM-associated protein 70) function in MEF proliferation and transformation is not well understood.
Purpose of the Study:
- To investigate the role of SWAP-70 in regulating MEF growth rates and spontaneous transformation.
- To determine if SWAP-70 acts as an oncogene or is required for transformation.
Main Methods:
- Cultivation and analysis of SWAP-70 deficient MEFs.
- Complementation of SWAP-70 deficiency by expressing human SWAP-70 in MEFs.
- Assessment of cell proliferation, contact inhibition, and transformation phenotypes.
- Inhibition studies using sanguinarine.
- Tumorigenicity assays in nude mice.
Main Results:
- SWAP-70 deficient MEFs failed to exhibit accelerated growth and maintained normal proliferation.
- Expression of human SWAP-70 in MEFs induced a transformation phenotype, including loss of contact inhibition and growth on top of each other.
- SWAP-70 expressing cells showed sustained activation of cdc2 and NFκB.
- Three out of four human SWAP-70 expressing MEF clones formed colonies in soft agar and/or tumors in nude mice.
- Mouse SWAP-70 also promoted MEF transformation.
Conclusions:
- SWAP-70 is essential for the spontaneous acceleration of MEF growth.
- SWAP-70 is required for, but not a typical oncogene driving, the spontaneous transformation of MEFs.
- Inhibition of SWAP-70 with sanguinarine reversed the transformed phenotype.
